<TPO and TPO Receptor>
Thrombopoietin (TPO) is a hematopoietic factor that promotes proliferation of megakaryocytes and platelets in vivo. Human TPO is a glycoprotein comprising 332 amino acid residues in full length, and the N-terminal sequence is known to be important for the activity of human TPO. Human TPO exhibits its functions upon binding to TPO receptor on cell membrane.
c-Mpl is the only TPO receptor that is known at present. Human c-Mpl is a glycoprotein having one transmembrane domain, comprising 635 amino acids if it contains signal peptide or 610 amino acids if it is matured, and it belongs to the type I cytokine receptor family. The messenger RNA and protein sequences of human c-Mpl have been already reported (Genbank: NM_005373, NP_005364). Examples of molecules of the same family include erythropoietin receptor (EpoR), G-CSF receptor (G-CSFR), and interleukin 3 receptor (IL-3R). Human c-Mpl has 2 CRH (cytokine receptor homologue) domains in its extracellular region (referred to as CRH1 and CRH2 from the N-terminus), and such domains comprise WSXWS (SEQ ID NO: 95) motif peculiar to the cytokine receptor family. The intracellular domain contains 2 sequences, Box1 and Box2, which are essential for signal transduction. It is suggested that TPO binds to CRH1 and dimerizes c-Mpl, thereby transducing a signal; however, specific modes of the binding and activation have not yet been elucidated. Upon dimerization of c-Mpl, signaling kinase that has bound to the intracellular domain is activated, and phosphorylation signal is transmitted within the cell. It is known that the TPO-Mpl signal activates Jak-STAT, PI3K-Akt, and Ras-MAPK pathways. In case of a mouse in which TPO or c-Mpl is defective, it is reported that the platelet count decreases to approximately 10%-20% relative to that of a wild-type mouse, indicating that the TPO-Mpl system is a critical system for regulating the platelet counts. c-Mpl expression is observed not only in megakaryocytes but also in undifferentiated hematopoietic progenitor cells or hematopoietic stem cells. c-Mpl-positive cell fractions in the bone marrow are known to have a higher ability to reconstruct bone marrow than c-Mpl-negative fractions. It is also known that a c-Mpl-deficient mouse has a decreased number of hematopoietic stem cells, as well as a decreased number of megakaryocytes and platelets (Hiroshi Miyazaki, “Future Prospects for Thrombopoietin,” Japanese Journal of Transfusion Medicine, 46(3) , 311-316, 2000; and Murone, M. et al., Stem Cell 16: 1-6, 1998). These findings suggest the involvement of the TPO-Mpl system with the hematopoietic system at the stem cell level or thereafter.
Since the cloning of TPO, its use as a therapeutic agent for thrombocytopenia has been expected, and clinical trials have been conducted in the past with respect to two types of recombinant TPOs: full-length human TPO (rhTPO) and PEG-rHuMGDF (pegylated recombinant human megakaryocyte proliferation and development factor) comprising a pegylated peptide sequence of the N-terminal 163 amino acids which form the active site of human TPO (Kuter, D J et al., Blood 100 (10): 3457-69, 2002). In the clinical trails, the recombinant TPOs were found to successfully increase platelets of healthy volunteers and patients with idiopathic thrombocytopenic purpura (ITP). Also, effects of reducing thrombocytopenia caused by nonmyeloablative chemotherapy have been demonstrated. Although the number of cases is small, effects of recombinant TPOs on patients with aplastic anemia (AA) or myelodysplastic syndrome (MDS) have been reported (Yonemura, Y. et al., Int J Hemat (82) 307-309, 2005; and Komatsu, N. et al., Blood 96, 296a, 2000).
<c-Mpl Agonist Antibody>
A variety of TPO mimetics having c-Mpl-mediating signaling properties as in TPO but having completely different molecular properties have been studied (Broudy, V C et al., Cytokine 25(2): 52-60, 2004; and Wang B. et al., Clin Pharmacol Ther., 76(6): 628-38, 2004). Known mimetics are roughly classified into, for example, peptidic lower molecules, nonpeptidic lower moleculest, antibody-derived molecules, agonist antibodies, and the like.
Examples of known anti-c-Mpl agonist human antibodies include 12B5, 12E10, and 12D5 (WO 99/10494). Such antibodies do not have activity against primary human cells in the form of a whole antibody, such as whole IgG. The term “primary human cell” as used herein refers to a cell on which TPO acts in vivo, such as CD34+ cell derived from human umbilical cord blood or bone marrow, but not an especially established cell line which is highly sensitive to TPO or a cell into which TPO receptor gene has been introduced and expressed at a high level. The term refers to cells on which TPO act in vivo, such as CD34+ cells derived from human umbilical cord blood or bone marrow.
Examples of known murine agonist antibodies include BAH-1 (WO 99/03495; and Deng B. et al., Blood 92(6): 1981-1988, 1998) and VB22B (WO 2005/056604). Murine antibodies are known to exhibit antigenicity in the human blood and thus are not appropriate as pharmaceuticals. In general, it is difficult to humanize agonist antibodies in the form of a whole antibody using, for example, CDR grafting while maintaining activity (WO 2005/056604; and Ji Hee Son et al., Journal of Immunological Methods 286: 187-201, 2004). Even if such known agonist antibodies are present accordingly, it is not easy to create agonist human antibodies that act on primary cells.
Antibody-derived lower molecules as described above in relation to the TPO mimetics are also represent a certain type of agonist antibody. Diabody and single chain (Fv)2 (sc(Fv)2) that are prepared by modifying part of an antibody have been reported (WO 99/10494; and WO 2005/056604). The modified antibodies that had been produced by such technique, however, may disadvantageously have antigenecity resulting from drastic modification of molecules. Also, their half-lives in blood would be shorter than that of the whole antibody. Thus, use of such modified antibodies as pharmaceuticals remains problematic.
Thus, the whole antibody has properties useful for pharmaceuticals, such as low antigenecity or half-life duration in blood; however, it is not easy to create agonist human antibodies having sufficient activity in the form of a whole antibody, as described above.
Accordingly, the present inventors have attempted to obtain agonist human human antibodies having sufficient activity without drastically modifying the antibody structure and, as a result, the present inventors have now succeeded in obtaining the antibodies of interest as described below. Moreover, the present inventors have now succeeded in modifying the hinge region of an antibody, thereby improving an agonist activity. Antibodies produced according to the present invention will be suitable as a therapeutic agent of thrombocytopenia.